Methods and compositions for reducing the environmental impact of animal waste

ABSTRACT

Methods for reducing the environmental impact of animal waste are described. In particular embodiments, the methods comprise administering to an animal an enzyme, such as alkaline phosphatase, that is effective to reduce the amount of a detrimental compound, such as ammonia or phosphorous, that is present in or released from animal waste. Also provided is a method for increasing phosphorus digestion in an animal. Compositions suitable for use in such methods are also described.

The present invention provides methods for reducing the environmentalimpact of animal waste. In particular, the invention provides methodscomprising administering to an animal an enzyme that is effective toreduce the amount of a detrimental compound present in or released fromanimal waste, and compositions suitable for use in such methods. Alsoprovided is a method for increasing phosphorus digestion in an animal.

Animal waste may contain or release one or more compounds that have adetrimental effect, such as a detrimental effect on the animal, on otheranimals, on humans, or on the environment. One such compound is ammonia(NH₃). Another such compound is phosphorous (P).

Atmospheric ammonia can have adverse effects on the environment, as wellas on animal production performance, health, and welfare. Ammoniageneration and emission in, for example, poultry housing, mostly resultfrom the microbiological decomposition of poultry waste. Ammonia levelsas low as 50 ppm can be detrimental to poultry, and such low levels maygo unnoticed. Exposure to ammonia at 50 ppm can contribute to 5-10% ofbirds being runts, and can be associated with a loss of 0.5 pounds ofmeat per bird and/or a loss of 8 points of feed conversion.

There is a need, therefore, for methods of reducing the amount of adetrimental compound present in or released from animal waste, such asfor reducing the ammonia and/or the phosphorous content of animal waste.

In accordance with some embodiments, there are provided methods forreducing the environmental impact of animal waste, comprisingadministering to an animal an effective amount of an enzyme that reducesthe amount of a detrimental compound present in or released from animalwaste. In accordance with some embodiments, there are provided methodsfor reducing the amount of ammonia in animal waste, comprisingadministering to an animal an effective amount of an enzyme that reducesthe amount of ammonia present in or released from animal waste. Inaccordance with some embodiments, there are provided methods forreducing the amount of phosphorous in animal waste, comprisingadministering to an animal an effective amount of an enzyme that reducesthe amount of phosphorous present in or released from animal waste. Alsoprovided is a method for increasing phosphorus digestion in an animal,comprising administering an effective amount of alkaline phosphatase tothe animal.

In accordance with any of these methods, the enzyme may be administeredorally.

In accordance with any of these methods, the enzyme may be alkalinephosphatase.

In accordance with any of these methods, the animal may be a poultry orswine animal.

In accordance with any of these methods, the enzyme may be administeredduring one or more of the starter phase, the grower phase, and/or thefinisher phase.

In accordance with any of these methods, the enzyme may be formulated inanimal feed, such as a starter feed, a grower feed, or a finisher feed.

In accordance with any of these methods, the enzyme may be formulated inan animal feed additive.

In accordance with some embodiments, there are provided compositionssuitable for oral administration to an animal, comprising an effectiveamount of an enzyme that reduces the amount of a detrimental compoundpresent in or released from animal waste. In accordance with someembodiments, there are provided compositions suitable for oraladministration to an animal, comprising an effective amount of an enzymethat reduces the amount of ammonia present in or released from animalwaste. In accordance with some embodiments, there are providedcompositions suitable for oral administration to an animal, comprisingan effective amount of an enzyme that reduces the amount of phosphorouspresent in or released from animal waste.

In accordance with any of these compositions, the composition maycomprise an orally acceptable carrier for the enzyme.

In accordance with any of these compositions, the enzyme may be alkalinephosphatase.

Any of these compositions may be suitable for administration to poultryor swine.

Any of these compositions may be an animal feed, such as a starter diet,a grower diet, or a finisher diet, or may be an animal feed additive.

As used in the following discussion, the terms “a” or “an” should beunderstood to encompass one or more, unless otherwise specified.

As used herein, the term “animal” refers to any animal, including humansand other animals, including companion animals such as dogs and cats,livestock, such as cows and other ruminants, buffalo, horses, swine(e.g., pigs or hogs), sheep, fowl or poultry (e.g., chicken, ducks,turkeys, and geese) and aquaculture animals (e.g., fish and shrimp andeels). A young animal is an animal which falls into the starter (orpre-starter) or grower category. Preferably, the young animal falls intothe starter (or pre-starter) category. For swine, an animal less than 25kilograms is also considered a young animal.

Described herein are methods comprising administering to an animal anenzyme that is effective to reduce the amount of a detrimental compoundpresent in or released from animal waste, such as ammonia (NH₃) orphosphorous (P), and compositions suitable for use in such methods. Themethods offer a number of advantages in the context of animalproduction, including poultry and swine production. For example, themethods may offer advantages such as reduced phosphate input into ananimal production system, decreased ammonia in animal manure, reducedventilation air requirements to dilute indoor ammonia concentration inanimal housing (and associated energy savings), and reduced need tofurther treat exhaust air.

While not wanting to be bound by any theory, the results reported belowindicate that the methods described herein may help animals (such asyoung broilers) utilize and digest the phosphorus that is present intheir diets, which in turn may lead to better growth rate and lessnutrient loss through excretion. Additionally or alternatively, themethods described herein may decrease NH₃ emission because the enzymetreatments may increase the metabolism and growth of favorable bacterialpopulations in the intestine, such that more of the excess nitrogen inthe diet remains in the manure as bacterial protein instead of uricacid, which is typically degraded and emitted as NH₃. Moreover, both thelower pH and lower nitrogen content in manure of treated animals maydeter and prevent the formation of gaseous NH₃ in the manure and reducethe NH₃ emission. The relationship between pH and degradation of uricacid (the major nitrogen source in poultry manure) has been reportedsuch that a sharp increase in pH may be associated with a decrease inthe uric acid content of poultry manure. Elliot & Collins, 1982,Transactions of ASAE 25: 413-24, indicated that high pH in the storedmanure would result in the majority of nitrogen loss as NH₃.Additionally, reducing the phosphorus content of animal waste may impactother properties of the manure, such as the bacterial flora.

In specific embodiments, the enzyme is alkaline phosphatase (AP) (EC3.1.3.1). Alkaline phosphatases occur in prokaryotic and eukaryoticorganisms, including mammals (including humans). For example, alkalinephosphatase is naturally present in breast milk and intestines, andplays a key role in digestion and digestion regulation. Alkalinephosphatase has been studied for use in therapeutic contexts (e.g., thetreatment of cancer, diabetes and weight loss). Comparison of theprimary structures of various alkaline phosphatases showed a high degreeof homology (25-30% homology between E. coli and mammalian). Millan,1988 Anticancer Res. 8, 995-1004; Harris, 1989 Clin. Chim. Acta 186,133-150. The alkaline phosphatase family includes the tissue-specificAPs (placental AP (PLAP), germ cell AP (GCAP) and intestinal AP (lAP>>and the non-tissue specific APs (TnAP) which are primarily located inthe liver, kidney and bones. U.S. Pat. No. 6,884,602 reports theexpression of alkaline phosphatase in yeast. Hundreds of microbialalakaline phosphatases have been described. See, e.g, BRENDA: TheComprehensive Enzyme Information System,http://www.brenda-enzvmes.orglphp/result flat.php4?ecno=3.1.3.1.Moreover, organisms can be engineered to produce enzymes with desiredproperties, such as increased activity. See, e.g., Du et al. J. Mol.Biol. 316: 941-53 (2002); Dealwis et al., Biochem. 34: 13967-73 (1995);Koutsioulis et al., Protein Eng'g Design & Selection 21: 319-27 (2008).

The invention also provides an alkaline phosphatase of SEQ ID NO:1, oran alkaline phosphatase having at least 70% sequence identity with SEQID NO:1. The following is SEQ ID NO:1:

VNKLLKGLAIGGIVLAVVSAGTLAVAKENASRAESSNGQSKNLIVLIGDGMGPAQVSAARYFQQHKNNINSLNLDPYYVGQATTYADRGEDGGHIVSGIVTSSASAGTAFATGNKTYNAAISVSNEDVSRPFASVLEAAELSGKSTGLVTTARITHATPAVYASHVRSRDNENAIAFQYLDSGIDVLLGGGESFFVTKEEKGKRNDKNLLPEFEAKGYKVVKTGQSLKSLSAKDAKVLGLFGGSHIAYVPDRSDETPSLAEMTSKALEILSTNENGFAIMIEGGRIDHAGHANDFPTMVQEALDFDEAFKVAIDFAKKDGNTSVVVTADHETGGLSLSRDNIYELNVDLWNKQKNSSESLVSALNEAKTIADVKKIVSDNTWITDLTNEEAQYILDGDGSSYKREGGYNAVISKRLLVGWSGHGHSAVDVGVWAYGPIADKVKGQIDNTRIATASAEVLGVDLKKATADLQSKYLYPKFKINRNKEVLFPAKPLAEALGGKYQAANGTATISGMSGTITVDLNAKKAKLSGNSSSITIDVDNDVLYLPLTAFSQITGQTLKWDALSERIMLKThe invention also provides alkaline phosphatases having at least 71,72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89,90, 91, 92, 93, 94 95, 96, 97, 98, or 99% sequence identity with SEQ IDNO:1. The invention also provides compositions containing at least oneof the above alkaline phosphatases, as well as methods of using such analkaline phosphatase for reducing the amount of one or more detrimentalcompounds present in or released from animal waste, increasing animalfeed conversion rate, increasing animal feed efficiency, and/orincreasing animal growth rate.

Sequence identity refers to a sequence that has a specified percentageof amino acid residues that are the same (i.e., share at least 70%identity, for example), when compared and aligned for maximumcorrespondence over a comparison window, or designated region asmeasured using a sequence comparison algorithms or by manual alignmentand visual inspection.

In specific embodiments, the methods comprise administering to an animalan amount of an enzyme, such as alkaline phosphatase, effective toreduce the amount of ammonia (NH₃) or phosphorous present in or releasedfrom the animal's waste. The amount may vary depending on the animal,the animal's diet, and other factors, and can readily be determined bythose skilled in the art using methods known in the art and illustratedin the examples. For example, the amount of ammonia (NH₃) and/orphosphorous present in or released from animal waste when given animalsare grown under given conditions can be measured and compared to thatpresent in or released from the animal waste of animals grown undercomparable conditions, but also administered an amount of the enzyme,such as alkaline phosphatase. (In this regard, it may be advantageous tocompare treated and control animals of the same age, as manureproperties may change with age, as discussed in the examples below). Adecrease in manure ammonia (NH₃) and/or phosphorous content or releaseassociated with administration of the enzyme indicates that an effectiveamount of enzyme was administered.

The enzyme typically is administered orally. However, the invention alsoencompasses embodiments where the enzyme is administered by other routesto the intestines or digestive tract, in accordance with knownpractices, such as via suppositories.

The enzyme may be provided in any form suitable for oral administration,such as liquid, solid, powder, gel, etc. The enzyme may be administeredalone, or may be formulated in any composition suitable for oraladministration. In some embodiments, the composition that is suitablefor oral administration is generally recognized as safe for oraladministration to an animal. For example, a composition that is suitablefor oral administration may contain only ingredients, and amounts ofsaid ingredients, that are generally recognized as safe for oraladministration to an animal, and does not contain any ingredients, oramounts of said ingredients, which are not generally recognized as safefor oral administration to an animal. Additionally or alternatively, acomposition that is suitable for oral administration contains onlyingredients, and amounts of said ingredients, that are allowed, or thatare not prohibited, for oral administration to an animal, and does notcontain any ingredients, or amounts of said ingredients, that are notallowed, or that are prohibited, for oral administration to an animal.

In some embodiments, the composition comprises an orally acceptablecarrier for the enzyme. As used herein, “orally acceptable carrier”includes any physiologically acceptable carrier suitable for oraladministration. Orally acceptable carriers include, without limitation,animal feed compositions, aqueous compositions, and liquid and solidcompositions suitable for use in animal feed products and/or for oraladministration to an animal, including liquid and solid animal feedadditives. Suitable carriers are known in the art, and include thosedescribed in U.S. Pat. No. 6,780,628.

In some embodiments, the composition is an animal feed. As used herein,the term “animal feed” has its conventional meaning in the field ofanimal husbandry. For example, animal feed includes edible materialswhich are consumed by livestock for their nutritional value. Animal feedincludes feed rations, e.g., compositions that meet an animal'snutritional requirements, and also include compositions that do not meetan animal's nutritional requirements. In some embodiments, the animalfeed is a starter feed, formulated for use during the starter period. Inother embodiments, the animal feed is a grower feed, formulated for useduring the grower period. In other embodiments the animal feed isfinisher feed used in the finishing period.

In specific examples of animal feed embodiment, the amount of enzyme(such as alkaline phosphatase) is at least about 10,000 internationalunits (IU) per U.S. ton of feed, at least about 15,000 internationalunits (IU) per U.S. ton of feed, at least about 20,000 internationalunits (IU) per U.S. ton of feed, at least about 25,000 internationalunits (IU) per U.S. ton of feed, at least about 30,000 internationalunits (IU) per U.S. ton of feed, at least about 35,000 internationalunits (IU) per U.S. ton of feed, at least about 40,000 internationalunits (IU) per U.S. ton of feed, at least about 45,000 internationalunits (IU) per U.S. ton of feed, at least about 50,000 internationalunits (IU) per U.S. ton of feed, at least about 60,000 IU per ton offeed, at least about 70,000 IU per ton of feed, at least about 80,000 IUper ton of feed, at least about 90,000 IU per ton of feed, at leastabout 100,000 IU per ton of feed, at least about 200,000 IU per ton offeed, at least about 500,000 IU per ton of feed, or at least about3,000,000 IU per ton of feed or higher.

In some embodiments, the amount of enzyme is in the range of about 25 toabout 75 MU/ton (MU=124,000 IU). In some embodiments, the amount ofenzyme is at least about 2 MU/ton (240,000 IU/ton or 264 IU/kg).

In other specific examples of animal feed embodiments, the amount ofenzyme (such as alkaline phosphatase) is at least about 10 IU/kg feed,at least about 15 IU/kg feed, at least about 20 IU/kg feed, such as atleast 20 IU/kg feed, at least at 25 IU/kg feed, at least 30 IU/kg feed,at least 35 IU/kg feed, at least at 40 IU/kg feed, at least at 45 IU/kgfeed, at least 50 IU/kg feed, at least 550 IU/kg, or more.

Thus, in some embodiments, the invention provides an animal feedcomprising an amount of an enzyme, such as alkaline phosphatase, that iseffective to reduce the amount of a detrimental compound, such asammonia (NH₃) and/or phosphorous, present in or released from animalwaste, and/or to increase digestion of phosphorus.

The feed composition may be prepared by methods known in the art. Forexample, the enzyme can be added to the other feed ingredients at anystage during the manufacturing process, as deemed to be appropriate bythose skilled in the art. In one embodiment, the enzyme is provided as asolution, such as a liquid enzyme concentrate that is added to otherfeed ingredients during the manufacturing process. Alternatively, anenzyme-containing solution is sprayed on to a substantially final formof the animal feed. In another embodiment, the enzyme is provided as asolid composition (such as a powder), such as a solid composition thatis added to other feed ingredients during the manufacturing process.Exemplary methods for manufacturing enzyme-containing feed are describedin WO 97/41739.

In some embodiments, the composition is other than an animal feed. Forexample, the composition may be a liquid composition other than ananimal feed or a solid composition other than an animal feed. Suchcompositions may be suitable for direct administration to an animal ormay be used as a feed additive (e.g., added to feed prior to feeding) ora feed supplement (including supplements that are diluted with otherfeed components prior to feeding and supplements that are offered to ananimal on a free choice, separate basis). Examples of a liquidcomposition other than an animal feed include liquid enzymeconcentrates, including liquid enzyme concentrates that are typicallydiluted or combined with other ingredients prior to oral administrationto an animal.

In embodiments where the composition is a liquid composition other thanan animal feed, such as an enzyme solution, the liquid composition orsolution may comprise enzyme (such as alkaline phosphatase) in an amountthat is at least about 40,000 international units (IU) per liter ofsolution, such as at least 40,000 IU/L, at least 50,000 IU/L, at least60,000 IU/L, at least 70,000 IU/L, at least 80,000 IU/L, at least 90,000IU/L, at least 100,000 IU/L, at least about 500,000 IU/L, at least about600,000 IU/L, at least about 700,000 IU/L, at least about 800,000 IU/L,at least about 900,000 IU/L, at least about 1,000,000 IU/L, at leastabout 2,000,000 IU/L, at least about 5,000,000 IU/L, or at least about200,000,000 IU/L.

In some embodiments, an amount of liquid composition other than ananimal feed, such as about 500 mL or 1000 mL solution, is applied to orcombined with an amount of feed, such as to a ton of feed, to arrive atfeed formulations with enzyme levels described above. In otherembodiments, an amount of liquid composition other than an animal feedis applied to or combined with an amount of feed to prepare an animalfeed with an amount of enzyme effective to reduce the amount of adetrimental compound, such as ammonia (NH₃) and/or phosphorous, presentin or released from animal waste, and/or to increase digestion ofphosphorus.

In embodiments where the composition is a solid composition other thanan animal feed, the composition may comprise enzyme (such as alkalinephosphatase) in an amount that is at least about 40,000 IU/kg, such asat least 40,000 IU/kg, at least 50,000 IU/kg, at least 60,000 IU/kg, atleast 70,000 IU/kg, at least 80,000 IU/kg, at least 90,000 IU/kg, atleast 100,000 IU/kg, at least 120,000 IU/kg, at least 140,000 IU/kg, atleast 160,000 IU/kg, at least 180,000 IU/kg, at least 200,000 IU/kg, orat least 60,000,000 IU/kg, or more.

In some embodiments, an amount of a solid composition other than ananimal feed is applied to or combined with an amount of feed to arriveat feed formulations with enzyme levels described above. In otherembodiments, an amount of solid composition other than an animal feed iscombined with an amount of feed to prepare an animal feed with an amountof enzyme effective to reduce the amount of a detrimental compound, suchas ammonia (NH₃) and/or phosphorous, present in or released from animalwaste, and/or to increase digestion of phosphorus.

In other embodiments, the enzyme is provided in a capsule or tablet formfor oral ingestion.

As conventional in the art, the term “IU” or “international unit” refersto an amount of enzyme that will catalyze the transformation of 1micromole of the substrate per minute under conditions that are optimalfor the enzyme. As used herein “MU” (Million Chemgen Units)=120,000 IU.(1 IU=8.33 ChemGen U) Weight equivalents for many enzymes are known inthe art and can be determined using standard assays. As known in theart, the selection of buffers and/or substrates can impact the unitsmeasured. Standard assays for alkaline phosphatase activity are known inthe art. See, e.g., Davidson, Enzyme Microb. Technol. 1: 9-14 (1979);Gonzalez-Gil et al., Marine Ecol. Prog. Ser. 164: 21-35 (1998);Sekiguchi et al., Enzyme Microb. Technol. 49: 171-76 (2011); Simpson etal., Promega Notes 74: 7-9.

In one embodiment of the invention, a dry composition of the inventionis present in an amount of more than 100 g per metric ton of completefeed. In one embodiment of the invention, a dry composition of theinvention is present in an amount of more than 500 g per metric ton ofcomplete feed.

In one embodiment of the invention, a dry composition of the inventionis present in an amount of between 10 g and 30 g per metric ton ofconcentrated premix. In one embodiment of the invention, a drycomposition of the invention is present in an amount of about 20 g permetric ton of concentrated premix.

In one embodiment of the invention, a liquid composition of theinvention is present in an amount of less than 100 ml per metric ton ofcomplete feed (liquid). In one embodiment of the invention, a liquidcomposition of the invention is present in an amount of 50-100 ml permetric ton of complete feed (liquid).

For use in any embodiment of the methods and compositions describedherein, the enzyme, such as alkaline phosphatase, can be obtained from acommercial source. Alternatively, the enzyme (including alkalinephosphatase) can be obtained from microorganisms that produce enzymes,such as bacteria, fungi and yeast.

Additionally, the enzyme can be obtained using recombinant technologymethods known in the art, by, for example, genetically engineering ahost cell to produce an enzyme, e.g., causing transcription andtranslations of a gene encoding the enzyme. Using known amino acidsequences or known nucleotide sequences encoding those sequences, thoseskilled in the art can design suitable genes for recombinant expressionof the enzyme. Additionally or alternatively, a nucleotide sequenceencoding a known enzyme, such as alkaline phosphatase, can be used toprobe a DNA library to identify other nucleotide sequences encodingenzymes suitable for use in the methods described herein. As known inthe art, such a DNA library can be derived from a defined organism orpopulation of organisms, or can be obtained from natural sources andthus represents DNA from microorganisms that are difficult to culture.

In any embodiment of the methods and compositions described herein, theenzyme, such as alkaline phosphatase, may be expressed by a plant thatis used in animal feed. For example, corn could be geneticallyengineered to express alkaline phosphatase and the resulting geneticallymodified com product could be used in feed. Production also can beeffected with other genetically modified or classically modified systemssuch as bacteria, e.g., E. coli, Bacillus sp., Lactobacillus; yeast,e.g., Pichia, Yarrow, Saccharomyces, Schizosaccharomyces (e.g.,Schizosaccharomyces pomb, Hansenula. Kluyveromyces, Candida), and otherfungus, such as Aspergillu, Rhizopus, Tricoderma, Humicola, Penicillium,and Humicola. In specific embodiments, the enzyme, such as alkalinephosphatase, is obtained from Bacillus lentus.

In embodiments where a composition comprises a combination of enzymes,the enzymes may be produced individually, by separate organisms, or twoor more of the enzymes may be produced by a single organism. Forexample, a single organism can be recombinantly engineered to producetwo or more enzymes by methods known in the art.

As noted above, the invention includes methods for reducing theenvironmental impact of animal waste, comprising administering to ananimal an effective amount of an enzyme that reduces the amount of adetrimental compound present in or released from animal waste. Theinvention also includes methods for reducing the amount of ammonia inanimal waste, comprising administering to an animal an effective amountof an enzyme that reduces the amount of ammonia present in or releasedfrom animal waste. The invention also includes methods for reducing theamount of phosphorous present in or released from animal waste,comprising administering to an animal an effective amount of an enzymethat reduces the amount of phosphorous present in animal waste. Theenzyme may be administered alone or in any composition described above,including an oral composition, such as animal feed, a liquid compositionother than an animal feed, or a solid composition other than an animalfeed. The animal may be any animal, including a human or a meatproduction animal, and may be a healthy animal or an animal sufferingfrom infection or other disease or condition.

In any of these methods, the enzyme may be administered orally, and maybe alkaline phosphatase.

In any of these methods, the animal may be a poultry animal, such aschickens, ducks, turkey, or geese, or a swine animal, such as pigs orhogs. In any of these methods, the enzyme may be administered during oneor more of the starter phase, the grower phase, and/or the finisherphase, or at any or all stages.

In any of these methods, the enzyme may be formulated in animal feed,including in a starter feed, a grower feed, or a finisher feed.Alternatively, in any of these methods, the enzyme may be formulated inan animal feed additive.

As noted above, the invention also includes compositions suitable fororal administration to an animal, comprising an effective amount of anenzyme that reduces the amount of a detrimental compound present in orreleased from animal waste. The invention also includes compositionssuitable for oral administration to an animal, comprising an effectiveamount of an enzyme that reduces the amount of ammonia present in orreleased from animal waste. The invention also includes compositionssuitable for oral administration to an animal, comprising an effectiveamount of an enzyme that reduces the amount of phosphorous present in orreleased from animal waste. In any of these compositions, thecomposition may comprise an orally acceptable carrier for the enzyme. Asnoted above, the effective amount of enzyme may vary from animal toanimal, and from enzyme to enzyme, but readily can be determined bythose skilled in the art, as described above and illustrated in Example3.

In any of these compositions, the enzyme may be alkaline phosphatase.

In any of these compositions, the composition may be suitable foradministration to poultry, such as chickens, ducks, turkey, or geese, orto swine, such as pigs or hogs.

In any of these compositions, the composition may be an animal feed,such as a starter feed diet or a grower feed diet. Alternatively, in anyof these compositions, the composition may be an animal feed additive.

In any embodiments of the invention, one or more additional activeingredients may be employed. An example of an additional activeingredient is another enzyme, which may have the same or differentproperties of the enzymes of the invention.

The following examples further illustrate the invention, but theinvention is not limited to the specifically exemplified embodiments.

EXAMPLE 1

Ammonia (NH₃) emissions of broiler manure samples from two feed additivetreatments (Mannanase HT-“HT” and Alkaline Phosphatase “AP”) wereevaluated during a 6-week grow-out period. The mannanase HT was added at60 MU/ton (240,000 IU/ton) and AP was added at an average of 141 MU/ton.The feed and water consumption, manure production and feed conversion ofthe broilers from three treatments (including control) were measured andreported. The nitrogen content, moisture content and pH of fresh manuresamples were analyzed. The experiment was conducted in emission vesselswith controlled air temperature and ventilation rate.

Feeding additives to broiler birds was shown to have the followingimpact on gaseous emissions and production performance:

(a) There was no significant difference in feed consumption, feedconversion, manure moisture content and pH observed for broilers fedwith HT or AP as compared to birds fed the control diet, although manurefrom the AP diet showed lower pH than manure from the control group.

(b) There was no difference in NH₃ emission rate (ER) and cumulativeemission between HT and control groups.

(c) The broilers fed the AP diet tended to have lower NH₃ emissions thanthe broilers fed control and HT diets.

(d) The NH₃ ER dramatically changed and increased exponentially from 28and 42 days.

(e) The efficacy of NH₃ ER by the AP diet tended to be age-dependentduring the three testing periods.

(t) The overall NH₃ ER rates for the 5 day storage periods were −7.4%from 35 day birds and 42.2% from 28 day birds in the AP group.

Materials and Methods

One hundred and seventeen 1-day-old female Ross 708 chicks were equallydistributed into three brooding houses (7.4 ft×7.4 ft, W×L). The birdsin each brooding house with a single pen were vaccinated with liveCoccidia vaccine at 4-day age and had access to water and one of thethree experimental diets (starter diet with HT, MT, or control). Thebrooding houses had the same temperature setup and lighting program.After 20-day age, 96 birds were transferred into grow-out cages in aventilated poultry house (9 ft×9 ft, W×L) and were housed in groups ofeight per cage (30 in×59 in, W×L).

Three grower diets were continuously fed to the birds for the grow-out.The eight birds in each cage were all from one of three treatments.After 35-day age, six birds were left in each cage to meet the animalwelfare standards. A total of twelve cages were randomly assigned tothree diets to minimize the location effect. The fresh manure from thebirds in each cage was collected with manure pans for NH3 emissionevaluation and analysis for five days on the days of 23, 28, 35, and 42.

Twelve 5-gallon (19-liter) emission vessels (EVs) were used to carry outthe evaluation. Twelve manure samples of each batch were collected and2.2 lb (1 kg) 2.2 lb (1 kg) manure samples were randomly placed in thetwelve EVs with 50 inch (324 cm²) surface area of each sample, andmeasured for the gaseous emissions over a 5 day period with airtemperature at 68° F. (20° C.) and air flow rate at 6.4 ft³/hr (3L/min). Both the air inlet and outlet were located in the air-tight lid.Teflon tubing (114 in or 0.635 cm diameter) was used in the emissionvessel system. The vessels were operated under positive pressure. Adiaphragm pump (Model DDL-80, Gast Inc., Benton Harbor, Mich.) was usedto supply fresh air to the emission vessels. Flow rate of the freshsupply air was be controlled and measured with an air mass flowcontroller (0 to 100 LPM, with stainless steel wetted parts, Aalborg,Orangeburg, N.Y.). The supply air was connected to a distributionmanifold where air was further divided via twelve identical flow meters(0.2 to 5 LPM, stainless steel valve, Dwyer Instruments, Inc., MichiganCity, Ind.). Each vessel was equipped with a small stirring fan (12 VDC)located 2 in (5 cm) below the lid for 1.5 for uniform mixing of theheadspace. Gas exhausted from the vessels was connected to a common 1.5in (3.75 cm) PVC pipe that was routed to the building vent outlet.Samples of the exhaust air from each of the twelve vessels and thesupply air were sequentially taken and analyzed at 5-minute intervals,with the first 3 min for stabilization and the last 2 min formeasurement. This yielded a measurement cycle of 65 min for each vessel.The sequential sampling was achieved by controlled operation of twelvesolenoid valves (Type 6014, 24V, stainless steel valve body. Burkert,Irvine, Calif.). The NH₃ concentration was measured with a photoacousticmulti-gas analyzer (INNOVA 1412, INNOVA, Denmark) that was challengedweekly and calibrated as needed with zero, and NH₃ calibration gases.Air temperature was measured with type T thermocouples (0.5° F.resolution). Analog outputs from the thermocouple, multi-gas analyzer,and the mass flow meter were sampled at 1-second interval and logged at1-minute intervals into a measurement and control unit (USC-2416,Measurement Computing Corp., Norton, Mass.).

Seven weeks were used to complete the emission measurements. Frozenmanure samples (0.25 lb/sample) were sent to a soil and water test labat University of Delaware for manure nutrient and property analysis,which includes Total Kjeldahl Nitrogen (TKN), ammoniacal nitrogen,moisture, and pH. Statistical analysis was performed using JMP 9.0 (SASInstitute, Inc., Cary, N.C.). The dietary effect was consideredsignificant at P-value ≦0.05.

Production Performances and Manure Properties

Production performances and fresh manure properties of the broilers fromthe three diets are shown in Tables 1 and 2.

TABLE 1 Manure properties (mean and standard error) of broiler birds fedwith three diets of Mannanase HT, B: Alkaline Phosphatase (AP), andCtrl: Control (n = 4). Bird MC*, NH₄ ⁺—N, NO₃ ⁻—N TKN, % age Trt^(#) % %(DM) (DM) (DM) pH 23-d A Mean 75.5 0.30 0.07 5.41 5.16 S.E. 1.1 0.060.01 0.35 0.07 B Mean 75.6 0.30 0.07 5.20 5.25 S.E. 0.7 0.04 0.02 0.280.05 Ctrl Mean 74.1 0.33 0.07 5.38 5.46 S.E. 0.4 0.02 0.03 0.16 0.2628-d A Mean 73.2 0.33 0.08 6.31 5.35 S.E. 2.2 0.03 0.03 0.30 0.11 B Mean73.6 0.35 0.04 5.52 5.31 S.E. 2.8 0.03 0.02 0.44 0.10 Ctrl Mean 72.90.42 0.12 5.77 5.40 S.E. 3.0 0.09 0.10 0.21 0.12 35-d A Mean 73.9 0.730.12 6.99 5.69 S.E. 1.0 0.10 0.05 0.29 0.07 B Mean 76.3 0.82 0.09 7.295.57 S.E. 0.9 0.08 0.03 0.23 0.17 Ctrl Mean 76.7 0.91 0.07 7.06 5.90S.E. 0.9 0.09 0.01 0.36 0.10 42-d A Mean 79.1 1.18 0.09 8.68 6.37 S.E.0.7 0.16 0.04 0.26 0.13 B Mean 79.5 1.61 0.03 8.41 6.38 S.E. 0.5 0.410.01 0.30 0.14 Ctrl Mean 78.5 1.44 0.07 7.66 6.55 S.E. 1.8 0.50 0.030.80 0.17 *A: Mannanase HT; B: Alkaline Phosphatase (AP); Ctrl: Control.*MC: manure moisture content; DM: dry matter basis; TKN: total kjedahlnitrogen; pH: fresh manure pH.

TABLE 2 Production performances (mean and standard error) of broilerbirds fed with three diets of A: Mannanase HT, B: Alkaline Phosphatase(AP), and Ctrl: Control (n = 4). Bird Manure*, Cumu feed*, Body*, ageTrt^(#) g bird⁻¹ d⁻¹ kg bird⁻¹ FCR* kg bird⁻¹ 23-d A Mean 66.0 1.07 1.440.74 S.E. 2.0 0.00 0.03 0.02 B Mean 65.9 1.12 1.45 0.77 S.E. 3.6 0.000.03 0.02 Ctrl Mean 64.9 1.10 1.38 0.80 S.E. 4.5 0.00 0.03 0.02 28-d AMean 147.0 1.87 1.53 1.23 S.E. 5.5 0.01 0.05 0.04 B Mean 145.6 1.93 1.591.22 S.E. 11.0 0.02 0.01 0.02 Ctrl Mean 135.3 1.88 1.56 1.21 S.E. 8.30.02 0.03 0.04 35-d A Mean 154.2 2.80 1.68 1.67 S.E. 12.8 0.02 0.03 0.02B Mean 178.4 2.88 1.68 1.72 S.E. 10.9 0.02 0.02 0.03 Ctrl Mean 179.82.80 1.65 1.70 S.E. 14.5 0.03 0.01 0.03 42-d A Mean 208.8 3.71 1.63 2.27S.E. 7.0 0.02 0.01 0.01 B Mean 234.2 3.79 1.67 2.27 S.E. 20.7 0.02 0.010.01 Ctrl Mean 216.4 3.72 1.64 2.28 S.E. 12.9 0.03 0.03 0.06 ^(#)A:Mannanase HT; B: Alkaline Phosphatase (AP); Ctrl: Control. *MC: manuremoisture content; Manure: fresh manure production rate; Cumu feed:cumulative feed consumption; FCR: feed conversion ratio; Body: bird bodyweight.

There was no significant difference among the three diets for productionperformances and manure properties (P at 0.05). The nitrogen contents(ammoniacal nitrogen: NH₄ ⁺-N and Total Kjeldahl Nitrogen: TKN) from thethree diets were at similar levels (P>0.28). There was a trend that theHT and AP diets had slightly lower manure pH during the grow-out. Thefresh manure of younger birds was more acidic than that from olderbirds.

Ammonia Emission and Reduction

NH₃ daily ER and cumulative emissions over a 5-day storage period forthe three diets are summarized and shown in Table 3.

TABLE 3 Mean (standard error) of ammonia daily emission rate (mg bird⁻¹d⁻¹) and cumulative emissions (mg bird⁻¹) from broiler manure during a5-d storage period (n = 4) with 10 air changes per hour (ACH) at 20° C.Daily ER, Cumulative Emission, mg bird⁻¹ d⁻¹ mg bird⁻¹ Storage time, dayAge Trt 1 2 3 4 5 1 2 3 4 5 23-d A Mean 0.07 0.53 3.16 6.72 8.50 0.070.61 3.77 10.5 19.0 S.E. 0.01 0.21 0.91 1.06 0.76 0.01 0.24 1.14 2.172.85 B Mean 0.06 0.26 1.29 3.20 5.25 0.06 0.32* 1.61* 4.81 10.1 S.E.0.00 0.07 0.41 1.07 1.40 0.00 0.07 0.48 1.57 3.00 Ctrl Mean 0.13 0.533.13 5.42 7.11 0.13 0.67 3.80 9.22 16.3 S.E. 0.02 0.13 0.83 0.95 0.880.00 0.07 0.47 1.55 2.96 28-d A Mean 0.26 1.23 5.10 10.9 17.4 0.26 1.496.60 17.5 34.9 S.E. 0.02 0.24 1.28 2.32 2.92 0.02 0.31 1.65 4.04 7.04 BMean 0.27 1.01 2.60 4.97* 10.2* 0.27 1.29 3.88 8.86 19.0 S.E. 0.04 0.221.27 2.58 3.10 0.03 0.29 1.61 4.29 7.42 Ctrl Mean 0.27 1.53 4.10 8.3718.6 0.27 1.79 5.90 14.3 32.9 S.E. 0.03 0.58 2.25 2.98 2.44 0.03 0.662.64 5.22 6.73 35-d A Mean 3.27 21.4 36.0 48.2 62.7 3.27 24.7 60.7 108.9171.6 S.E. 1.30 5.91 7.85 9.68 13.7 1.21 7.47 15.2 25.1 38.9 B Mean 2.7018.2 36.2 52.0 68.7 2.70 20.9 57.1 109.1 177.9 S.E. 0.79 3.59 7.64 14.522.8 0.75 4.66 12.7 28.2 51.2 CM Mean 9.82 33.9 37.6 39.3 44.8 9.82 43.781.4 120.7 165.6 S.E. 3.31 4.08 1.92 1.91 2.18 3.35 7.29 8.91 9.9 10.742-d A Mean 13.8 46.9 66.9 77.1 81.4 13.8 60.7 127.6 204.7 286.1 S.E.3.62 6.80 6.61 6.61 10.8 2.04 3.66 10.7 25.4 40.3 B Mean 15.1 43.8 47.957.3 62.3 15.1 58.9 106.8 164.1 226.4 S.E. 4.50 7.82 6.66 8.81 11.4 4.579.91 22.1 41.1 62.3 Ctrl Mean 23.0 48.2 60.9 63.3 63.7 23.0 71.2 132.1195.4 259.1 S.E. 9.84 11.0 9.51 9.67 11.5 10.1 20.8 30.0 38.2 47.1The NH₃ ERs were the lowest over the 5-day storage period for the APdiet at 23 and 28 days. There was no difference on NH₃ ER and cumulativeemission between HT and control diets. There were some significantdifferences between the AP and control diets (P<0.05). For instance, theNH₃ ERs on the 4th and 5th day from 28-day birds were significantlylower (P=0.02). The NH₃ ER dramatically changed and increasedexponentially from 28 and 42 days. For example, the ER from 35-day birdson the 2nd day of the 5-day storage was 18.2 mg/bird/day, which was muchhigher than the ER (1.01 mg/bird/day) from 28-day birds. This changecould be related to the increasing trend of manure pH from less than 5.5to 6.5. The ammoniacal N is in the form of NH₄ ⁺, which is not volatilewith low pH.

The efficacy of NH₃ emission reduction by the AP diet tended to beage-dependent during the testing period. The efficacy of NH₃ emissionreduction by the AP diet decreased with increasing bird age (Table 4).

TABLE 4 Reduction rate (percentage) of cumulative ammonia emissionsduring a 5-d storage period (n = 4) Bird Storage time, day Treatment age1 2 3 4 5 A 23-d 46.8 9.4 0.9 −13.8 −16.3 28-d 1.7 16.8 −11.9 −22.7 −6.035-d 66.7 43.5 25.4 9.8 −3.6 42-d 39.8 14.7 3.4 −4.8 −10.4 B 23-d 56.852.9 57.7 47.9 38.4 28-d −2.1 28.2 34.1 37.9 42.2 35-d 72.6 52.2 29.89.6 −7.4 42-d 34.2 17.2 19.1 16.0 12.6In comparison, NH₃ ER reduction varied from 57.7% from 23-day birds, to19.1% from 42-day birds for the AP diet after storage time. The overallNH₃ emission reduction rates for the 5-day period were −7.4% from 35-daybirds and 42.2% from 28-day birds for the AP regimens.

The outcome of the variable dietary efficacy could have stemmed fromchanges in manure properties, especially moisture content and pH, as themicrobial activities varied considerably. Under the productioncondition, the broiler houses with young birds tend to have higher NH₃concentration at bird level due to limited ventilation, conservingheating energy, and lowering gas usage. Lowering NH₃ emissions frombroiler houses with young birds will help to establish healthy flocksand reduce the risk of disease breakout related to high NH₃concentration.

EXAMPLE 2

In this study, ammonia (NH₃) emissions of broiler chickens fed with anenzyme feed additive (Alkaline Phosphatase—AP) were evaluated during a30-day grow-out period. The broilers were brooded on wood shavings until12-days of age and moved into cages for rest of the grow-out. Freshmanure was collected on days 14, 22, and 30, and tested for NH₃emissions in emission vessels with controlled air temperature andventilation rate. The feed and water consumption, manure production, andfeed conversion of the broilers were measured and reported. The nutrientcontent, moisture, and pH of the fresh manure samples were analyzed.Feeding enzyme additives to broiler birds was shown to have thefollowing impact on gaseous emissions and production performance:

(a) AP diet improved broiler growth and feed conversion ratio at ages of22- and 30 days.(b) AP diet reduced manure pH at 22-days and lowered phosphorus by 6 and7% at 14- and 22-days.(c) NH₃ emission was significantly reduced by 23.8% over a 4-day periodat 14-days.(d) Overall NH₃ emission reduction rates for the 4-day period were 5.9%from 30-day birds and 30.7% from 22-day birds.

Materials and Methods

Four temperature controlled houses (7.4 ft×7.4 ft, W×L) with new woodshavings were used for brooding. Each brooding house was equipped withone 1.5 kW space heater and 150 W heat lamp with one cup drinker (1 ftdiameter) and one feeder (1 ft diameter). Two groups (3-days apart) of148 day-old female Ross 708 female chicks from a same breeder flock werecollected and equally distributed into the two brooding houses (74 birdsper house). The birds in each brooding house with a single pen werevaccinated with live Coccidia vaccine and had access to water and twoexperimental diets (AP and control) with the AP added at 60 MU/ton toboth starter and grower diets. The brooding houses had identicaltemperature and lighting programs. The birds were fed ad libitum withtwo diets: control and AP. After 12 days, groups of 12 birds per cagewere transferred to grow-out cages (30 in×30 in, W×L) in two identicalhouses (9 ft×9 ft, W×L), 12 cages per house. Each cage had two nippledrinkers, one trough drinker (2.5 in×30 in, W×L), and one trough feeder(5 in×30 in, W×L). The bird numbers were reduced to 8 birds per cage on22-day age. A total of 24 cages were assigned to the two houses byrandom block design to minimize the house effect. The birds in the samehouse were of the same age. The light program was 23:1 hour (Light:Dark)for the brooding period and 24 hour light for the rest of grow-out. Thetemperature of each house was measured and recorded by a temperaturelogger (HOBO U23, Onset Comp., Pocasset, Mass.) with 5 minute intervals.The birds were weighed on days 0, 12, 14, 22, and 30. The feed usage wasrecorded daily and feed conversion ratio (FCR) was calculated. Thestarter diet was as follows:

INGREDIENT AMOUNT Corn US #2 1000 Soybean Meal Hi Pro 513 48% CP Meat ML55/Blend 52 110 DDGS 160 Bakery Dex 60 Canola ML 50 SUB-TOTAL 1893 CornMicro Flush 18.05 Limestone 13.00 Dicalcium Phosphate 11.00 18.5% PBioLys 50% 7.90 DL-Methionine 5.54 S-Carb-30 3.00 Salt 1.20 HyD 83.30.60 Choline Chloride 60% 1.52 Tmin + EDDI 1.25 Vitamins 2X 0.60 QuantumPhytase 2500 D 0.44 Mintrex-Cu 0.40 Poultry Fat Pet Food 42.50 GradeTOTAL BATCH 2000.00 WEIGHTThe grower diet was as follows:

INGREDIENT AMOUNT Corn US #2 1000 Soybean Meal Hi Pro 513 48% CP Meat ML55/Blend 52 110 DDGS 160 Bakery Dex 60 Canola ML 50 SUB-TOTAL 1893 CornMicro Flush 18.05 Limestone 13.00 Dicalcium Phosphate 11.00 18.5% PBioLys 50% 7.90 DL-Methionine 5.54 S-Carb-30 3.00 Salt 1.20 HyD 83.30.60 Choline Chloride 60% 1.52 Tmin + EDDI 1.25 Vitamins 2X 0.60 QuantumPhytase 2500 D 0.44 Mintrex-Cu 0.40 Poultry Fat Pet Food 42.50 GradeTOTAL BATCH 2000.00 WEIGHT

Stainless steel manure pans were used to collect the fresh manure fromthe cages in Block 1 on days 12, 20, and 28, and from the cages in Block2 on days 13, 21, and 29, for a 2-day period. Twelve 2.2 lb (1 kg)manure samples were taken for NH₃ emission test on days 14, 22, and 30for Block 1, and days 15, 23, and 31 for Block 2, respectively.Additional 0.5 lb samples were stored in a −20° C. freezer and sent to acertified lab (Midwest Laboratories, Omaha, Nebr.) for nutrient andproperty analysis, which included total Kjeldahl nitrogen (TKN), ammonianitrogen (NH₃—N), phosphorus (reported as P₂O₅), potassium (K₂O),sulfur(S), moisture, and pH.

Twelve 5-gallon (19-liter) emission vessels (EVs) were used to carry outthe NH₃ emission evaluation. Twelve manure samples of each block werecollected and 2.2-lb manure samples were randomly placed in the twelveEVs with 50 inch (324 cm²) surface area of each sample, and measured forthe gaseous emissions over a 4-day period with air temperature at 75° F.(24 C) and air flow rate at 6.4 ft³/hr (3 L/min). Both the air inlet andoutlet were located in the air-tight lid. Teflon tubing (0.635 cm or ¼in. diameter) was used in the emission vessel system. The vessels wereoperated under positive pressure. A diaphragm pump (Model DDL-80, GastInc., Benton Harbor, Mich.) was used to supply fresh air to the emissionvessels. Flow rate of the fresh supply air was be controlled andmeasured with an air mass flow controller (0 to 100 LPM, with stainlesssteel wetted parts, Aalborg, Orangeburg, N.Y.). The supply air wasconnected to a distribution manifold where air was further divided viatwelve identical flow meters (0.2 to 5 LPM, stainless steel valve, DwyerInstruments, Inc., Michigan City, Ind.). Each vessel was equipped with asmall stirring fan (12 VDC) located 2 in (5 cm) below the lid foruniform mixing of the head space. Gas exhausted from the vessels wasconnected to a common 1.5 in (3.75 cm) PVC pipe that was routed to thebuilding vent outlet. Samples of the exhaust air from each of the twelvevessels, the supply air and the ambient air were sequentially taken andanalyzed at 5 minute intervals, with the first 3 minutes forstabilization and the last 2 minutes for measurement. This yielded ameasurement cycle of 65 minutes for each vessel. The sequential samplingwas achieved by controlled operation of twelve solenoid valves (Type6014, 24V, stainless steel valve body, Burkert, Irvine, Calif.). The NH₃concentration was measured with a photoacoustic multi-gas analyzer(INNOVA 1412, INNOVA, Denmark) that was challenged weekly and calibratedas needed with zero, and NH₃ calibration gases. Air temperature wasmeasured with type T thermocouples (0.5° F. resolution). Analog outputsfrom the thermocouple, multi-gas analyzer, and the mass flow meter weresampled at 1-s intervals and logged at 1-min intervals into ameasurement and control unit (USB-2416, Measurement Computing Corp.,Norton, Mass.).

The data from the two blocks at the similar ages were pooled andanalyzed for the dietary and age effect. Therefore, three age groups at14-, 22-, and 30-days were used to represent 14- to 15-day, 22- to23-day, and 30- to 31-day, respectively. Statistical analysis wasperformed using JMP 9.0 (SAS Institute, Inc., Cary, N.C.). The dietaryeffect was considered significant at P-value ≦0.05.

Production Performances and Manure Properties

Production performances of the broilers from the two diets are shown inTable 5.

TABLE 5 Production performances and manure properties (mean and standarderror) of broiler birds fed with two diets of Ctrl: Control, and Trt:Alkaline Phosphatase (AP) (n = 12). Bird MC*, DM, Manure*, Cumu feed*,Body*, kg age Trt^(#) % g bird⁻¹ d⁻¹ g bird⁻¹ d⁻¹ kg bird⁻¹ FCR* bird⁻¹14-d Ctrl Mean 72.0 16.82 60.2 0.58 1.35 0.43 S.E. 0.93 0.78 2.40 0.020.05 0.01 Trt Mean 73.0 17.49 65.7 0.60 1.35 0.44 S.E. 0.95 0.55 3.150.01 0.01 0.00 22-d Ctrl Mean 73.3 24.5 92.5 1.24 1.42 0.86 S.E. 1.140.94 3.74 0.03 0.04 0.01 Trt Mean 72.6 24.2 88.9 1.27 1.45 0.88 S.E.0.95 0.67 2.79 0.02 0.02 0.01 30-d Ctrl Mean 77.6 33.1 149.8 2.02 1.361.48 S.E. 0.73 0.71 6.43 0.01 0.01 0.01 Trt Mean 77.5 34.1 153.5 2.011.31 1.54 S.E. 0.86 0.73 6.10 0.01 0.02 0.02 ^(#)Ctrl: Control; Trt:Alkaline Phosphatase (AP). *MC: moisture content; DM: dry manureproduction rate Manure: fresh manure production rate; Cumu feed:cumulative feed consumption; FCR: feed conversion ratio; Body: bird bodyweight.There was no significant difference from AP diet on manure productionrate at either age. The birds with AP diet had better growth rate atages of 22- and 30-day (P=0.037 and 0.006). At 30-day age, the AP grouphad better FCR (1.31 vs. 1.36) than the control group (P=0.03).

The manure properties of the two diets are presented in Table 6.

TABLE 6 Nutrient content (percentage of dry matter basis) and pH (meanand standard error) of fresh manure from two diets of Ctrl: Control, andTrt: Alkaline Phosphatase (AP) (n = 12). Bird NH₃—N*, TKN*, P₂O₅*, K₂O*,S*, age Trt^(#) pH % % % % % 14-d Ctrl Mean 5.77 0.25 5.29 2.36 2.910.62 S.E. 0.05 0.01 0.32 0.06 0.06 0.02 Trt Mean 5.73 0.22 5.09 2.22^(a)2.89 0.62 S.E. 0.13 0.01 0.22 0.04 0.04 0.01 22-d Ctrl Mean 6.37 0.375.46 2.27 2.84 0.61 S.E. 0.13 0.03 0.24 0.05 0.05 0.01 Trt Mean 5.98^(a)0.30 5.34 2.12^(a) 2.91 0.60 S.E. 0.08 0.02 0.15 0.04 0.04 0.01 30-dCtrl Mean 6.25 0.75 5.50 2.20 2.81 0.60 S.E. 0.15 0.07 0.23 0.05 0.080.01 Trt Mean 6.44 0.69 5.61 2.17 2.74 0.59 S.E. 0.17 0.07 0.19 0.060.07 0.01 ^(#)Ctrl: Control; Trt: Alkaline Phosphatase (AP). *NH₃—N:ammonia nitrogen; TKN: total Kjeldahl nitrogen; S: sulfur.It shows that the AP diet reduced manure pH at 22-d (P=0.05) and loweredphosphorus (2.22 vs. 2.36% and 2.12 vs. 2.27%) at 14-day (P=0.05) and22-day (P=0.02). In addition, there is a trend that AP diet could causeless NH₃—N and TKN in the manure.

These results indicate that the AP diet could help young broilersutilize and digest the nitrogen and phosphorus, which leads to bettergrowth rate and less nutrient loss through excretion. Both lower pH andnitrogen content in manure could deter and prevent the formation ofgaseous NH₃ in the manure and reduce the NH₃ emission.

Ammonia Emissions

NH₃ daily emission rate (ER) and cumulative emissions over a 4-dayperiod for the two diets are summarized and shown in Table 7.

TABLE 7 Mean (standard error) of ammonia daily emission rate (mg bird⁻¹d⁻¹) and cumulative emissions (mg bird⁻¹) from broiler manure during a4-d storage period (n = 12) with 10 air changes per hour (ACH) at 24° C.Daily ER, Cumulative Emission, mg bird⁻¹ d⁻¹ mg bird⁻¹ Storage time, dayAge Trt 1 2 3 4 1 2 3 4 14-d Ctrl Mean 0.82 3.30 5.21 5.99 0.51 3.719.64 13.6 S.E. 0.24 0.30 0.55 0.59 0.06 0.36 0.92 1.33 Trt Mean 0.672.59* 3.88* 4.48* 0.45 2.95* 7.43* 10.4** S.E. 0.18 0.17 0.36 0.47 0.050.32 0.59 0.86 22-d Ctrl Mean 10.7 17.1 20.8 21.5 6.17 23.3 51.7 68.2S.E. 1.98 3.93 4.26 3.70 2.13 4.73 10.2 13.2 Trt Mean 6.88 11.8 15.015.7 3.84 15.4 35.4 47.3 S.E. 1.96 2.26 2.19 1.97 1.23 3.68 6.99 8.5930-d Ctrl Mean 19.2 21.5 22.2 22.4 19.6 47.8 79.3 95.2 S.E. 3.30 1.532.18 2.52 5.47 8.33 8.86 8.83 Trt Mean 17.6 19.5 21.7 22.3 18.9 44.474.0 89.6 S.E. 4.98 2.90 1.75 2.24 7.41 12.6 14.0 13.4The NH₃ ERs and cumulative emissions were significantly reduced over the4-day storage period for the AP diet at 14-day (p≦0.04). The cumulativeNH₃ emission of broiler manure decreased 23.8% after 4-day storage bythe AP diet. Daily emission rate had the similar decreasing trend. Therewas no difference on NH₃ ER and cumulative emission between the twodiets at 22- and 30-day due to large variations. However, there istendency that the AP diet still reduces the ER and cumulative emissionat 22 day (P=0.1).

The reduction rate of cumulative emissions with AP diet ranged from 31to 38% at 22-d (Table 8).

TABLE 8 Reduction rate (percentage) of cumulative ammonia emissionsduring a 4-d storage period (n = 12) Storage time, day Bird age 1 2 3 414-d 13.3 20.8 23.0 23.8 22-d 37.8 33.9 31.7 30.7 30-d 3.95 7.24 6.735.89The efficacy of NH₃ emission reduction by the AP diet was age dependentduring the testing period. The birds age significantly affect the NH₃emission due to lower nitrogen content and pH in the manure of youngerbirds (P<0.01). This change could be related to the increasing trend ofmanure pH from 5.73 to 6.44. The ammoniacal N is in the form of NH₄ ⁺,which is not volatile with low pH. Temperature also plays an importantrole on the NH₃ emissions since microbial activity and NH₃volatilization are directly impacted by temperature. The outcome of thevariable dietary efficacy could have stemmed from changes in manureproperties, especially nitrogen content and pH, as the microbialactivities varied considerably.

Under production conditions, the broiler houses with young birds tend tohave higher NH₃ concentration at bird level due to limited ventilation.Lowering NH₃ emissions from broiler houses with young birds will help toestablish healthy flocks and reduce the risk of disease breakout relatedto high NH₃.

EXAMPLE 3

This study is conducted to demonstrate that diets containing alkalinephosphatase added at 36 MU/ton in the treated group can reduce the NH₃emissions under commercial conditions relative to a non-AP treatedcontrol, by verifying the efficacy of the additive selected from Example2.

This field verification test was conducted using one house measuring 48m×13.5 m (160 ft×45 ft) which is divided into 16 partitions, 6 m×6 m (20ft×20 ft) each. The 6 rooms used in this study were managed separately,but share the same bird genetics and production stage, allowing bettercomparison the six flocks of birds with two different diets. Three roomsheld flocks randomly signed with control diet, while the otherscontained birds with AP diet. Broilers over a 38-d growout period wereraised in the house with control or treatment randomly assigned tohouse. Each room had an initial placement of 530 straight-run birds(mixed sex, Cobb×Cobb) with new wood shaving. The production rooms haveinsulated ceilings, box air inlets along the central alley, one broodingheater (30,000 BTU), one 0.3-m (12-in) centrifugal fan and one 0.6-m(24-in) diameter fan located on the side wall of the house. Independentenvironmental controllers coordinate control of air temperature,ventilation fan and heater operation, and lighting programs. Airtemperature was monitored by a temperature sensor and logger (TMC6 andU12, Onset, Pocasset, Mass.).

Production performance data for both control and treatment rooms,including feed consumption, body weight, feed efficiency, and birdmortality, were collected. Bird live weight of each room was measuredand recorded by a bird scale. The birds were fed and mortality wasrecorded daily. Three phase feeding strategy was used: starter from dayzero to 13-d, grower from 14- to 31-d, and finisher from 32- to 38-d.The feed added into each room was weighed and recorded. At the end ofthe trial, the birds were weighed again and feed conversion ratio (FCR)was calculated.

A multi-point air sampling (Pak III, CAI, Orange, Calif.) and dataacquisition system (SCADA3000, Sensaphone, Aston, Pa.) were used tomonitor the control (Ctrl) and treatment (Trt) rooms with a 5-secinterval. The ON/OFF status of each fan was monitored by a AC-DC relay.The room static pressure was measured with a differential pressuresensor (T-VER-PXU-L, Onset, Pocasset, Mass.). A photoacoustic multi-gasanalyzer (Innova Model 1412, CAI, Orange, Calif.) was used to measurethe concentrations of NH₃, CO₂ and dew point temperature of the exhaustair. The Innova 1412 analyzer has been shown to be highly accurate,stable and responsive. Exhaust air samples from the fans in each housewere drawn and analyzed to ensure good representation of the house airbeing exhausted to the atmosphere. The sampling port was placed betweenthe two fans, 1.8 m (6 ft) above the floor and two feet apart from thewall. The air sampling interval was set to 140 sec per room (5samples/room×28 sec/sample). In addition to the gaseous concentrations,the corresponding building ventilation rate will be measured by in-situcalibration of the exhaust fans with a FANS unit and continuousmonitoring of operational state of the fans and the room static pressure(Equation 1).

VR=a+b×SP  [1]

where

-   -   VR=fan ventilation rate, m³/hr;    -   a, b=fan curve coefficients; and    -   SP=static pressure, Pa.

Three litter samples were taken from each room for nutrient and chemicalanalysis, including ammonia nitrogen (NH₃—N), total kjeldahl nitrogen(TKN), moisture content, and pH. NH₃ emission rate was calculated withconcentration and ventilation rate [Equation 2].

ER=VR/n×(C _(e) −C _(i))×(17.031 g/mol)/(22.414 L/mol)  [2]

where

-   -   ER=emission rate, mg/bird-hr;    -   VR=ventilation rate, m³/hr;    -   C_(e)=exhaust NH₃ concentration, ppmv;    -   C_(i)=inlet NH₃ concentration, ppmv; and    -   n=bird number per room.        Daily NH₃ emission rate (ER) and cumulative emissions over the        38-d growout period for the two diets were calculated and used        for the data analysis. The data from each diet were pooled and        analyzed for the dietary and age effect. Statistical analysis        was performed using JMP 9.0 (SAS Institute, Inc., Cary, N.C.).        The significance of dietary effect was indicated as ** with        P-value ≦0.05 and * with P-value ≦0.1.

Production Performances and Manure Properties

Production performances of the broilers from the two diets are shown inTable 9.

TABLE 9 BW, Livability, NH₃—N, g/bird FCR % % TKN, % MC, % pH Ctrl 24001.70 92.4 1.05 3.62 31.6 7.1 Trt 2421 1.72 91.4 1.13 3.29 33.2 7.3 * BW:marketed bird body weight; FCR: feed conversion ratio; MC: moisturecontent; TKN: total Kjeldahl nitrogen.There was no significant difference between the two diets on body weightgain and livability. The birds with AP diet had better growth rate atages of 32-d (P=0.08). However, the control birds had lower FCR than thetreatment group (P=0.002). There was a clear trend that the growth curveof the treatment dropped which may be caused by the changing feed after31-d of age. It shows that there is no difference between the two dietson NH₃—N, TKN, pH, and moisture content at the end of the 38-dmonitoring.

Ammonia Emissions

Daily NH₃ emission rate (ER) and cumulative emissions over the 38-dgrowout period for the two diets were summarized and shown in Table 10.

TABLE 10 ER, mg/bird-d Cumulative Emission, g/bird Bird Mean s.e Means.e. age Ctrl Trt Ctrl Trt Ctrl Trt Ctrl Trt 7 8.71 5.40 1.2 1.2 0.0670.038 0.01 0.01 8 11.2 6.59 1.6 1.0 0.079 0.045 0.01 0.01 9 9.03 5.01 .20.7 0.089 0.050 0.01 0.01 10 7.30 3.91 1.3 0.4 0.10 0.054 0.01 0.01 1111.0 7.29 1.1 0.8 0.11 0.062 0.01 0.01 12 10.9 8.81 1.3 1.3 0.12** 0.0720.01 0.01 13 12.7 9.10 1.7 0.5 0.13** 0.081 0.02 0.01 14 16.7 11.8 3.20.5 0.15** 0.094 0.02 0.01 15 17.1* 11.3 2.3 0.8 0.17** 0.11 0.02 0.0116 20.1 17.1 1.8 1.8 0.19** 0.12 0.02 0.01 17 15.5* 9.07 2.5 1.0 0.21**0.13 0.02 0.01 18 22.5 14.9 3.8 0.9 0.23** 0.15 0.03 0.01 19 20.2 17.43.1 7.0 0.25** 0.17 0.03 0.02 20 45.0 39.4 5.8 0.2 0.30** 0.21 0.03 0.0221 52.8 34.1 8.9 2.2 0.36** 0.25 0.04 0.02 22 152 148 9.3 2.7 0.52**0.40 0.04 0.02 23 201 156 19.4 11.1 0.73** 0.57 0.06 0.03 24 272 21539.7 16.9 1.01** 0.79 0.10 0.03 25 395 327 49.2 35.3 1.42** 1.14 0.140.05 26 227 179 21.4 21.3 1.66** 1.32 0.16 0.06 27 292 263 20.1 39.31.96** 1.59 0.18 0.10 28 391 405 6.2 35.4 2.36* 2.01 0.18 0.12 29 384340 25.9 49.6 2.76* 2.36 0.20 0.17 30 500 514 31.1 62.9 3.27* 2.89 0.220.23 31 529 525 36.6 46.7 3.81 3.43 0.25 0.27 32 543 571 27.4 54.5 4.364.01 0.27 0.32 33 528 573 37.6 37.1 4.89 4.59 0.28 0.36 34 602 659 11.031.5 5.50 5.26 0.29 0.39 35 616 638 20.2 27.4 6.12 5.90 0.31 0.40 36 621518 59.1 8.0 6.75 6.42 0.35 0.41 37 622 574 11.5 16.4 7.37 7.00 0.360.40 38 343 354 20.1 57.7 7.72 7.35 0.37 0.38 *P ≦ 0.1; **P ≦ 0.05.The NH₃ emissions of AP diet were significantly lower than the controldiet till 28-d at P≦0.05 level and 31-d at P≦0.1 level. The NH₃ emissionreduction rate decreased from 40% at 12-d of age to 19% at 27-d of ageand 10% at 31-d of age. Daily emission rate had the similar decreasingtrend. The field test shows the similar results that the efficacy of NH₃emission reduction by the AP diet was age-dependent in the previous labstudy (Li, 2011). The bird age significantly affected the NH₃ emissiondue to lower nitrogen content and pH in the manure of younger birds.Under the production condition, litter will be reused the broiler houseswith young birds tend to have higher NH₃ concentration at bird level dueto limited ventilation. Lowering NH₃ emissions from broiler houses withyoung birds will reduce bird level NH₃ concentration and reduce the riskof disease breakout related to poor air quality with high NH₃concentration.

Based on Examples 2 and 3, feeding AP additives to broiler birds wasshown to have the following impact on NH₃ emissions and productionperformance^(.)

1) No significant difference on bird body weight, livability, and litterproperties was observed from AP diet during the 38-d grow out. The birdswith AP diet had better growth rate. However, the control group hadbetter feed conversion ratio.

2) AP diet reduced manure pH (P=0.05) at 22-d and lowered phosphorus by6 and 7% at 14- and 22-d (P=0.02). There was a trend that AP diet couldreduce NH₃—N and TKN in the manure.

3) NH₃ emission from fresh manure was significantly reduced by 23.8%over a 4-d period with AP diet at 14-d. The efficacy of NH₃ emissionreduction by the AP diet was age-dependent during the three testingperiod. The overall NH₃ emission reduction rates for the 4-d period were5.9% from 30-d birds and 30.7% from 22-d birds.

4) The efficacy of NH₃ emission reduction by the AP diet under the fieldcondition was age-dependent during the 38-d grow out period. The NH₃emission was significantly reduced by 19% at 27-d of age and 10% at 31-dof age. The overall NH₃ emission reduction rates for the 38-d periodwere 4.7%.

5) These results indicate that the AP diet could help young broilersutilize and digest the nitrogen and phosphorus, which leads to bettergrowth rate and less nutrient loss through excretion. Both lower pH andnitrogen content in manure could deter and prevent the formation ofgaseous NH₃ in the manure and reduce the NH₃ emission.

EXAMPLE 4

A dose-escalation trial was conducted in turkeys. The experiment wasdesigned as a randomized block design, with four dietary treatments(control and three different levels of alkaline phosphatase) randomlyassigned among 4 blocks of 12 battery cages (pens) each. Turkeys werefed these diets ad libitum until 4 weeks. On day 24 of age of the birds,manure was collected. The manure was analyzed as described in Examples 1and 2 above.

The test diets included alkaline phosphatase at four different levels:A: 0 (control); B: 22 MU/ton feed; C: 49 MU/ton feed; and D: 74 MU/tonfeed (1 MU=120,000 IU). The diet was a typical starter diet supplementedwith nutrients, as shown below.

Description Starter diet Ingredients Percent CARGILSBM 42.414 CORN 201034.741 DDGS 6.000 POULTRY MEAL 5.000 POULTRY FAT 3.990 DICALP 18.5 2.959Celite 2.000 LIMESTONE 1.200 ALIMET 0.403 MICRO SALT 0.322 LYSINE 0.316CHOLINE CHLORIDE 60% 0.192 NCSU TRACE MINERAL PREMIX² 0.200 NCSU VITAMINPREMIX² 0.150 SODIUM SELENITE PREMIX¹ 0.050 L-THREONINE 0.063 Total %100.00 ¹NaSeO3 premix provided 0.3 mg Se/kg of complete feed ²Eachkilogram of mineral premix (.1% inclusion) supplied the following per kgof complete feed: 60 mg Zn as ZnS0₄H₂0; 60 mg Mn as MnS0₄H₂0; 40 mg Feas FeS0₄H₂0; 5 mg Cu as CuS0₄; 1.25 mg I as Ca(I0₃)₂; 1 mg Co as CoS0₄.³Each kilogram ofvitamin premix (.1% inclusion) supplied the followingper kg of complete feed: vitamin A, 13,200 IU; cholecalciferol, 4,000IU; alpha-tocopherol, 66 IU; niacin, 110 mg; pantothenic acid, 22 mg;riboflavin, 13.2 mg; pyridoxine, 8 mg; menadione, 4 mg; folic acid, 2.2mg; thiamin, 4 mg; biotin, 0.253 mg; vitamin B12, 0.04 mg; ethoxyquin,100 mg.

Nutrients Unit Weight KG 1.0000 DRY MATTER, % PCT 90.6418 ME POULTRY,KCALIKG KCAL/KG 2950 CRUDE PROTEIN, % PCT 28.50 MOISTURE, % PCT 9.36CRUDE FAT, % PCT 6.88 CRUDE FIBRE, % PCT 2.59 CALCIUM, % PCT 1.45 TOTALPHOSPHORUS, % PCT 1.062 AVAIL. PHOS. POULTRY PCT 0.800 SODIUM, % PCT0.180 POTASSIUM, % PCT 1.290 CHLORIDE, % PCT 0.324 Na + K—Cl, MEQ/KGMEQ/K,G 317.23 ARGININE, % PCT 1.9236 DIG. ARG TURKEY, % PCT 1.8287LYSINE, % PCT 1.8500 DIG. LYS TURKEY, % PCT 1.6924 METHIONINE, % PCT0.8165 DIG. MET TURKEY, % PCT 0.7390 MET + CYS, % PCT 1.2500 DIG. TSAATURKEY, % PCT 0.9767 THREONINE, % PCT 1.1500 DIG. THR TURKEY, % PCT0.9487 TRYPTOPHAN, % PCT 0.3244 DIG. TRP POULTRY, % PCT 0.2820 CHOLINE,MG/KG MG/KG 2720.0000

The data show a dose-response relationship across diets A, B, and D. Theresults achieved with Diet C did not fit the dose-response relationship,but still showed reduced NH₃ ER as compared to control.

Manure Properties

There was no significant difference among the three AP diets for manureproperties (P at 0.05). However, there was a trend that the B, C, and Ddiets with AP had lower manure pH and TKN, which could change themetabolism and growth of bacterial populations and cause less NH₃formation and volatilization. The fresh manure from D diet had thelowest pH of 5.83 compared to 6.37, 6.15, and 6.38 from A, B, and Cdiets, respectively. The TKN of B, C, and D were 2.45, 2.38, and 2.57%,which were lower than 3.61% from A diet.

Ammonia Emission and Reduction

The NH₃ ERs of D diet were the lowest over the 7-d storage period. Therewas no difference on NH₃ ER and cumulative emission among the four diets(P>0.05) due to large variation. The large variation of NH₃ emissionscould be caused by the uneven moisture content of the manure samples.The manure moisture contents of all 48 samples varied for 54 to 83%.Although the dietary effect is not significant, the results stillreveals that the NH₃ emission could be reduced while the mean emissionvalues were compared. The overall NH₃ ER reduction varied from 6.5 to40% for the B (20.6%), C (6.5%), and D (40%) diets compared to A dietafter the 7-d storage time. The outcome of the variable dietary efficacycould have stemmed from changes in manure properties, especiallymoisture content and pH, as the microbial activities variedconsiderably. Under the commercial production condition, the largevariation of NH₃ emission among the manure with different moisture levelcould be cancelled out. It suggested that the manure samples should bemore uniform and manure sample collection should be more carefullycarried out.

In summary, feeding additives to turkeys was shown to have the followingimpact on ammonia emission and manure properties:

1) Manure on B, C, and D diets (with AP) showed lower pH and totalnitrogen content than the A diet (control).

2) There was no significant difference on NH₃ ER and cumulative emissionamong the four diets due to large variation caused by big range ofmanure moisture content. The turkeys with B, C, and D diets (AP) tendedto have lower NH₃ emissions than the A diet (control).

3) The overall NH₃ emission reduction rates for the 7-d period were−6.5% from C diet and 40% for D diet.

EXAMPLE 5

The transition of a nursing pig to dry feed at weaning is a verystressful time in the life of the pig. In addition to adapting to a newdietary regime, the pig must adjust to a new group feeding structure andthe potential disease challenge to its immune system.

Another challenge for the pork producer is environmental regulationscurrently focusing on whole farm nutrient balance especially onnutrients that can have an impact on water quality. Phosphorus, inparticular, is a nutrient that is excreted in manure in excessiveamounts and has become the limiting nutrient dictating how much manurecan be applied to cropland. Normal corn contains P but onlyapproximately 15% of the P is digestible when fed to the pig. Therefore,inorganic P must be added to the diet to meet the pig's requirements forP, and the non-digestible P in the diet is excreted.

There is a needed to evaluate the value of alkaline phosphatase innursery pig diets based on pig growth, feed intake, feed efficiency andhealth. In addition, the availability of phosphorus in nursery pig dietsand the amount of P excreted needs to be determined as the pig's gutdevelops post-weaning and diets continue to change. The objective ofthis study is to determine the nutritional and environmental value ofalkaline phosphatase in nursery pig diets based on nutrientdigestibility and excretion.

A metabolism trial is conducted to evaluate nutrient digestibility andexcretion from nursery pigs fed alkaline phosphatase supplemented diets.Twelve barrows are used with six barrows per dietary treatment. Pigs arefed a phase 1 pelleted conventional corn-soy-whey based diet for 7 dayspost-weaning. Pigs are then blocked by weight and ancestry to one of twodietary treatments.

Pigs are housed in pairs in the metabolism pens for the first 6 days ofthe metabolism period, adjusting to the phase 2 experimental diets fedtwice daily at near ad libitum before being split into individualmetabolism crates and fed restricted intake for 4 days prior toinitiating a 3-day total urine and fecal collection. During collection,pigs are housed in individual stainless steel metabolism crates(2.33×2.83 ft.) fitted with nipple waterers. Screens and collection pansare placed beneath the crates to allow for separate and total urine andfeces collection. Urine collection buckets are acidified with 100 mL 10%HCl to prevent ammonia volatilization.

The 4 days prior and during the 3 day collection period, pigs are fedtwice daily at 07:30 and 17:00 at a rate of 9% of BW^(0.75). Eachmorning of the collection, total orts, urine, and feces are collected,measured, and frozen at −20 C for later analysis. Daily samples fromeach pig are pooled for later analysis. Feces are thawed and blendedwith deionized water to make a 50:50 by weight slurry that is blendedand then sub-sampled with 1 sample kept as a fecal slurry for theNitrogen assays and a sample that iss freeze dried and was used for allother nutrient assays.

Diets are ground through a 1 mm screen in a Wiley Mill (ThomasScientific, Swedesboro, N.J.) prior to analysis. Dry matter isdetermined following a 16 h drying period at 100° C. Total N (Nelson andSommers, 1972) and AmmN (Bremner and Keeney, 1965) are determined bymicro-Kjeldahl procedures. Total P are determined colormetrically(Murphy and Riley, 1962) using a Beckman Du-6 Spectrophotometer (BeckmanCoulter, Irvine, Calif.). Diet, fecal, and urine energies are determinedby bomb calorimeter. Urine energy is determined by drying 4 mL of urineon sulka floc in the energy capsule and then combusting the sulka flocand urine in the bomb calorimeter and then determining the urinaryenergy by difference from the sulka floc treated with deionized water.

Pig was the experimental unit. The GLM procedure of SAS is used todetermine statistical differences between treatments. Statisticalsignificances are indicated by P<0.05 while statistical trends areindicated by P<0.10. Two experimental diets were used during themetabolism study: 1) Control, and 2) Control +60 MU AP. These dietsallowed for a comparison between conventional and AP supplemented diets,and are noted below.

Phase 2 Metabolism Diets

Control Control + 60 MU AP Ingredient, % Corn 40.485 40.485 SBM, 48% CP22.330 22.330 Swine Grease 1.000 1.000 Limestone 0.420 0.420 MonocalciumPhosphate 0.300 0.300 Vitamin Premix 0.250 0.250 Trace Mineral Premix0.150 0.150 Selenium Premix 0.050 0.050 Salt 0.300 0.300 Plasma Protein2.500 2.500 SD Blood Meal 2.000 2.000 Fish Meal 4.000 4.000 PoultryMeal-NRC 4.000 4.000 Dried Whey 20.000 20.000 Lysine-HCL 0.100 0.100DL-Methionine 0.170 0.170 L-Threonine 0.070 0.070 Carbadox-2.5 1.0001.000 Chromic Oxide 0.375 0.375 Corn TRT Premix 0.500 0.500 Total100.000 100.000 Calculated Nutrients ME, kcal/kg 3295.10 3295.10 CP, %25.48 25.48 Total Lysine, % 1.680 1.680 Digestible Lysine, % 1.427 1.427SID Lysine, % 1.500 1.500 Dig. Methionine, % 0.504 0.504 Dig. Met + Cys,% 0.835 0.835 Dig. Threonine, % 0.894 0.894 Dig. Tryptophan, % 0.2470.247 Dig. Isoleucine, % 0.821 0.821 Dig. Valine, % 1.038 1.038 Ca, %0.850 0.850 P, % 0.743 0.743 Avail. P, % 0.500 0.500 AnalyzedComposition CP, % 23.5 23.7 P, % 0.57 0.61 Gross Energy, kcal/kg 39753987 Dry matter, % 88.54 88.47

Results are provided in Table 11, entitled Effect of Alkalinephosphatase (AP) enzyme during the Early Nursery period on Thy Matter(DM), Energy, Nitrogen (N), and Phosphorus (P) digestibility andexcretion.

TABLE 11 Probability, 0 60 SE P< AP, MU/ton Initial BW, lb 16.38 16.370.343 0.97 Day 6 BW, lb 19.57 19.63 0.484 0.92 Final BW, lb 24.05 24.530.522 0.53 ADG d 0-6, lb/d 0.531 0.544 0.054 0.86 ADG d 6-13 lb/d 0.6400.700 0.025 0.12 ADG d 0-13 lb/d 0.590 0.628 0.029 0.38 Total collectiond10-13 DM offered, g/d 413.8 413.5 7.50 0.98 DM intake, g/d 372.4 398.912.91 0.18 Feces DM, g/d 54.8 58.0 4.16 0.60 Urine, mL/d 551.4 631.983.46 0.51 DM, % digested 85.4 85.5 0.87 0.94 Energy intake, kcal/d1480.3 1590.9 51.30 0.16 Fecal Energy, kcal/d 259.6 275.3 20.00 0.59Urinary Energy, kcal/d 139.6 157.5 21.49 0.57 Energy digested, % 82.5782.68 1.033 0.94 Energy retained, % 72.73 72.77 1.426 0.99 Total NIntake, g/d 14.0 15.2 0.49 0.13 Feces, g/d 2.95 3.05 0.205 0.74 Urine,g/d 1.51 1.97 0.272 0.26 N, % digested 79.10 79.91 1.007 0.58 NRetained, % intake 68.33 66.84 2.140 0.63 NH₄—N Feces, g/d 0.48 0.440.036 0.37 P Intake, g/d 2.13 2.45 0.075 0.01 Feces, g/d 1.05 1.05 0.0510.98 Urine, g/d 0.024 0.010 0.0122 0.42 P, % digested 50.53 57.14 1.8050.03 Retained, % intake 49.23 56.75 1.900 0.02

There was no effect of diet on growth rate of the pigs during the firstweek of the diets being fed. However, during the second week (d 6-13),pigs fed the alkaline phosphatase tended (P<0.12) to grow slightlyfaster (9.4%) than the unsupplemented pigs (Table 11).

Alkaline phosphatase did not affect DM, Energy, or Nitrogendigestibility or excretion (P>0.26). However, due to the slightly higherfeed intake and analyzed dietary phosphorus, pigs fed the AP diet hadhigher phosphorus intake (P<0.01), but similar fecal and urinaryexcretion of phosphorus. This resulted in pigs fed AP to have greaterphosphorus digestibility (P<0.03) and retention (P<0.02) than pigs fedthe control diet.

While not being bound by theory, there may be opportunity for the APenzyme to improve the gut physiology of the weaned pig and this may leadto more efficient utilization of phosphorus.

We claim:
 1. A method for reducing the environmental impact of animal waste, comprising administering to an animal an effective amount of an enzyme that reduces the amount of one or more detrimental compounds present in or released from animal waste.
 2. The method of claim 1 wherein said detrimental compound is ammonia.
 3. The method of claim 1 wherein said detrimental compound is phosphorous.
 4. The method of any of claims 1 to 3, wherein said enzyme is administered orally.
 5. The method of any of claims 1 to 4, wherein said enzyme is alkaline phosphatase.
 6. The method of any of claims 1 to 5, wherein said enzyme is alkaline phosphatase, and said alkaline phosphatase is the following SEQ ID NO:1, or an alkaline phosphatase having at least 70% structural identity to SEQ ID NO:1: VNKLLKGLAIGGIVLAVVSAGTLAVAKENASRAESSNGQSKNLIVLIGDG MGPAQVSAARYFQQHKNNINSLNLDPYYVGQATTYADRGEDGGHIVSGIV TSSASAGTAFATGNKTYNAAISVSNEDVSRPFASVLEAAELSGKSTGLVT TARITHATPAVYASHVRSRDNENAIAFQYLDSGIDVLLGGGESFFVTKEE KGKRNDKNLLPEFEAKGYKVVKTGQSLKSLSAKDAKVLGLFGGSHIAYVP DRSDETPSLAEMTSKALEILSTNENGFAIMIEGGRIDHAGHANDFPTMVQ EALDFDEAFKVAIDFAKKDGNTSVVVTADHETGGLSLSRDNIYELNVDLW NKQKNSSESLVSALNEAKTIADVKKIVSDNTWITDLTNEEAQYILDGDGS SYKREGGYNAVISKRLLVGWSGHGHSAVDVGVWAYGPIADKVKGQIDNTR IATASAEVLGVDLKKATADLQSKYLYPKFKINRNKEVLFPAKPLAEALGG KYQAANGTATISGMSGTITVDLNAKKAKLSGNSSSITIDVDNDVLYLPLT AFSQITGQTLKWDALSERIMLK


7. The method of any of claims 1 to 6, wherein said animal is a young animal.
 8. The method of any of claims 1 to 7, wherein said animal is a chicken or turkey.
 9. The method of any of claims 1 to 7, wherein said animal is a swine.
 10. The method of any of claims 1 to 9, wherein the enzyme is administered during one or more of the starter phase, the grower phase, and/or the finisher phase.
 11. The method of any of claims 1 to 10, wherein said enzyme is formulated in animal feed.
 12. The method of any of claims 1 to 10, wherein said enzyme is formulated in an animal feed additive.
 13. The method of any of claims 1 to 12, wherein one or more additional active ingredients are administered to said animal.
 14. A composition suitable for oral administration to an animal comprising an effective amount of an enzyme that reduces the amount of one or more detrimental compounds present in or released from animal waste, and one or more orally acceptable carriers.
 15. The composition of claim 14 wherein said detrimental compound is ammonia or phosphorous.
 16. The composition of claim 14 or 15, wherein said enzyme is alkaline phosphatase.
 17. The composition of any of claims 14 to 16, wherein said enzyme is alkaline phosphatase, and said alkaline phosphatase is the following SEQ ID NO:1, or an alkaline phosphatase having at least 70% sequence identity to SEQ ID NO:1: VNKLLKGLAIGGIVLAVVSAGTLAVAKENASRAESSNGQSKNLIVLIGDG MGPAQVSAARYFQQHKNNINSLNLDPYYVGQATTYADRGEDGGHIVSGIV TSSASAGTAFATGNKTYNAAISVSNEDVSRPFASVLEAAELSGKSTGLVT TARITHATPAVYASHVRSRDNENAIAFQYLDSGIDVLLGGGESFFVTKEE KGKRNDKNLLPEFEAKGYKVVKTGQSLKSLSAKDAKVLGLFGGSHIAYVP DRSDETPSLAEMTSKALEILSTNENGFAIMIEGGRIDHAGHANDFPTMVQ EALDFDEAFKVAIDFAKKDGNTSVVVTADHETGGLSLSRDNIYELNVDLW NKQKNSSESLVSALNEAKTIADVKKIVSDNTWITDLTNEEAQYILDGDGS SYKREGGYNAVISKRLLVGWSGHGHSAVDVGVWAYGPIADKVKGQIDNTR IATASAEVLGVDLKKATADLQSKYLYPKFKINRNKEVLFPAKPLAEALGG KYQAANGTATISGMSGTITVDLNAKKAKLSGNSSSITIDVDNDVLYLPLT AFSQITGQTLKWDALSERIMLK


18. The composition of any of claims 14 to 17, wherein said animal is a young animal.
 19. The composition of any of claims 14 to 18, wherein said animal is a chicken or turkey.
 20. The composition of any of claims 14 to 18, wherein said animal is a swine.
 21. The composition of any of claims 14 to 20, wherein the enzyme is adapted to be administered during one or more of the starter phase, the grower phase, and/or the finisher phase.
 22. The composition of any of claims 14 to 21, wherein said enzyme is formulated in animal feed.
 23. The composition of any of claims 14 to 21, wherein said enzyme is formulated in an animal feed additive.
 24. The composition of any of claims 14 to 23, wherein said composition comprises one or more additional active ingredients.
 25. A method for increasing phosphorus digestion in an animal, comprising administering to said animal an effective amount of alkaline phosphatase to increase digestion of phosphorus by said animal.
 26. The method of claim 25 wherein said alkaline phosphatase is the following SEQ ID NO:1, or an alkaline phosphatase having at least 70% structural identity to SEQ ID NO:1: VNKLLKGLAIGGIVLAVVSAGTLAVAKENASRAESSNGQSKNLIVLIGDG MGPAQVSAARYFQQHKNNINSLNLDPYYVGQATTYADRGEDGGHIVSGIV TSSASAGTAFATGNKTYNAAISVSNEDVSRPFASVLEAAELSGKSTGLVT TARITHATPAVYASHVRSRDNENAIAFQYLDSGIDVLLGGGESFFVTKEE KGKRNDKNLLPEFEAKGYKVVKTGQSLKSLSAKDAKVLGLFGGSHIAYVP DRSDETPSLAEMTSKALEILSTNENGFAIMIEGGRIDHAGHANDFPTMVQ EALDFDEAFKVAIDFAKKDGNTSVVVTADHETGGLSLSRDNIYELNVDLW NKQKNSSESLVSALNEAKTIADVKKIVSDNTWITDLTNEEAQYILDGDGS SYKREGGYNAVISKRLLVGWSGHGHSAVDVGVWAYGPIADKVKGQIDNTR IATASAEVLGVDLKKATADLQSKYLYPKFKINRNKEVLFPAKPLAEALGG KYQAANGTATISGMSGTITVDLNAKKAKLSGNSSSITIDVDNDVLYLPLT AFSQITGQTLKWDALSERIMLK


27. The method of claim 25 or claim 26, wherein said alkaline phosphatase is administered orally.
 28. The method of any of claims 25 to 27, wherein said animal is a young animal.
 29. The method of any of claims 25 to 28, wherein said animal is a swine.
 30. The method of any of claims 25 to 29, wherein said alkaline phosphatase is administered during one or more of the starter phase, the grower phase, and/or the finisher phase.
 31. The method of any of claims 25 to 30, wherein said alkaline phosphatase is formulated in animal feed.
 32. The method of any of claims 25 to 31, wherein said alkaline phosphatase is formulated in an animal feed additive.
 33. The method of any of claims 25 to 32, wherein one or more additional active ingredients are administered to said animal.
 34. An alkaline phosphatase of SEQ ID NO:1, or an alkaline phosphatase having at least 70% sequence identity to SEQ ID NO:1: VNKLLKGLAIGGIVLAVVSAGTLAVAKENASRAESSNGQSKNLIVLIGDG MGPAQVSAARYFQQHKNNINSLNLDPYYVGQATTYADRGEDGGHIVSGIV TSSASAGTAFATGNKTYNAAISVSNEDVSRPFASVLEAAELSGKSTGLVT TARITHATPAVYASHVRSRDNENAIAFQYLDSGIDVLLGGGESFFVTKEE KGKRNDKNLLPEFEAKGYKVVKTGQSLKSLSAKDAKVLGLFGGSHIAYVP DRSDETPSLAEMTSKALEILSTNENGFAIMIEGGRIDHAGHANDFPTMVQ EALDFDEAFKVAIDFAKKDGNTSVVVTADHETGGLSLSRDNIYELNVDLW NKQKNSSESLVSALNEAKTIADVKKIVSDNTWITDLTNEEAQYILDGDGS SYKREGGYNAVISKRLLVGWSGHGHSAVDVGVWAYGPIADKVKGQIDNTR IATASAEVLGVDLKKATADLQSKYLYPKFKINRNKEVLFPAKPLAEALGG KYQAANGTATISGMSGTITVDLNAKKAKLSGNSSSITIDVDNDVLYLPLT AFSQITGQTLKWDALSERIMLK


35. The alkaline phosphatase of claim 34, wherein said enzyme has at least 70% sequence identity to SEQ ID NO:1.
 36. The alkaline phosphatase of claim 34 or 35, wherein said enzyme has at least 90% sequence identity to SEQ ID NO:1.
 37. The alkaline phosphatase of any of claims 34 to 36, wherein said enzyme has at least 95% sequence identity to SEQ ID NO:1.
 38. The alkaline phosphatase of any of claims 34 to 37, wherein said enzyme has at least 99% sequence identity to SEQ ID NO:1.
 39. A composition comprising an alkaline phosphatase of any of claims 34 to 38 and one or more acceptable carriers.
 40. The composition of claim 39, wherein it further comprises at least one additional active ingredient.
 41. Use of an alkaline phosphatase of any of claims 34 to 38 for reducing the amount of one or more detrimental compounds present in or released from animal waste, increasing animal feed conversion rate, increasing animal feed efficiency, and/or increasing animal growth rate.
 42. An alkaline phosphatase of any of claims 34 to 38 for use in therapy.
 43. An alkaline phosphatase of any of claims 34-38, for use in reducing the amount of one or more detrimental compounds present in or released from animal waste, increasing animal feed conversion rate, increasing animal feed efficiency, increasing phosphorus digestion, and/or increasing animal growth rate. 